Effect of activin receptor-like kinase 5 (ALK5) knockdown (KD) on transforming growth factor (TGF)-β1-induced migration (chemokinesis) in Panc1-RAC1B-KD and knockout (KO) cells. (A) Panc1 cells were transfected twice with either 50 nM of control siRNA, 25 nM RAC1B siRNA+ 25 nM control siRNA, or 25 nM RAC1B siRNA + 25 nM ALK5 siRNA. Forty-eight hours after the second transfection, the cells were processed for migration assay on the xCELLigence platform. Immediately before the start of the assay, one-half of the cells received 5 ng/mL TGF-β1. (B) As in (A) except that Panc1-RAC1B-KO cells received 50 nM of either siCo or siALK5. Data in (A) and (B) are from one representative experiment and are the mean ±SD from 3–4 wells per condition. Differences between Panc1 + RAC1B siRNA + ALK5 siRNA + TGF-β1 (black curve, tracing C) and Panc1 + RAC1B siRNA + TGF-β1 (magenta curve, tracing B) are significant at 16:30 and all later time points, while in (B) those between Panc1-RAC1B-KO + ALK5 siRNA + TGF-β1 (black curve, tracing C) and Panc1-RAC1B-KO + control siRNA + TGF-β1 (magenta curve, tracing B) are significant at 07:00 and all later time points. Successful inhibition of RAC1B and ALK5 was verified by immunoblotting (data not shown). For functional validation of the ALK5 siRNA, see Figure 1A.